Adaptive Landscapes of Plasmodium Falciparum Dihydrofolate Reductase Reveal Pathways to Antifolate Resistance

Abstract Antifolate resistance in Plasmodium falciparum dihydrofolate reductase (pfDHFR) remains a major challenge for malaria control. To understand how this enzyme maintains function under antifolate selection, we developed PfPATH, a computational framework that integrates mutational fitness measurements, evolutionary interactions, and structural information to map adaptive trajectories. PfPATH adaptive walks reveal that evolution is constrained to a narrow ridge in sequence space defined by a small subset of residues. Most resistance mutations are individually deleterious and become viable only when supported by stabilizing changes, restricting adaptation to a few high-resistance pathways. Structure network analyses show that these mutations do not disrupt the global enzyme architecture but instead reorganize internal communication to preserve catalytic function. Consistent with this, molecular dynamics and free-energy analyses indicate that resistant variants remain stable while sampling a broader ensemble of low-energy conformational substates. Together, these results reveal a narrow and predictable route to antifolate escape.